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1.
BMC Microbiol ; 20(1): 200, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32640994

RESUMO

BACKGROUND: Rickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation. RESULTS: Three hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera. CONCLUSION: Our proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteômica/métodos , Rickettsia akari/isolamento & purificação , Rickettsiose do Grupo da Febre Maculosa/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Cromatografia Líquida , Humanos , Modelos Moleculares , Peso Molecular , Estrutura Secundária de Proteína , Coelhos , Rickettsia akari/imunologia , Rickettsia akari/metabolismo , Rickettsiose do Grupo da Febre Maculosa/imunologia , Espectrometria de Massas em Tandem
2.
Int J Mol Sci ; 21(10)2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32414116

RESUMO

Bread wheat (Triticum aestivum L.) is one of the most valuable cereal crops for human consumption. Its grain storage proteins define bread quality, though they may cause food intolerances or allergies in susceptible individuals. Herein, we discovered a diversity of grain proteins in three Ukrainian wheat cultivars: Sotnytsia, Panna (both modern selection), and Ukrainka (landrace). Firstly, proteins were isolated with a detergent-containing buffer that allowed extraction of various groups of storage proteins (glutenins, gliadins, globulins, and albumins); secondly, the proteome was profiled by the two-dimensional gel electrophoresis. Using multi-enzymatic digestion, we identified 49 differentially accumulated proteins. Parallel ultrahigh-performance liquid chromatography separation followed by direct mass spectrometry quantification complemented the results. Principal component analysis confirmed that differences among genotypes were a major source of variation. Non-gluten fraction better discriminated bread wheat cultivars. Various accumulation of clinically relevant plant proteins highlighted one of the modern genotypes as a promising donor for the breeding of hypoallergenic cereals.


Assuntos
Albuminas/genética , Proteínas de Grãos/química , Proteoma/genética , Triticum/genética , Albuminas/química , Albuminas/metabolismo , Pão/análise , Grão Comestível/química , Grão Comestível/genética , Eletroforese em Gel Bidimensional , Gliadina/química , Gliadina/genética , Globulinas/química , Globulinas/genética , Glutens/química , Glutens/genética , Proteínas de Grãos/classificação , Humanos , Triticum/química
3.
Int J Mol Sci ; 21(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198416

RESUMO

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.


Assuntos
Alanina/química , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Animais , Catálise , Hipóxia Celular , Movimento Celular , Cães , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Células Madin Darby de Rim Canino , Espectrometria de Massas , Neoplasias/metabolismo , Prognóstico , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Espectrometria de Massas em Tandem
4.
J Cell Mol Med ; 23(5): 3641-3655, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30916466

RESUMO

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.


Assuntos
Anidrase Carbônica I/genética , Exossomos/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Interferência de RNA , Anidrase Carbônica I/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Metabolismo Energético/genética , Exossomos/metabolismo , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
5.
Ecotoxicol Environ Saf ; 173: 86-95, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30769207

RESUMO

Large areas polluted with toxic heavy metals or radionuclides were formed as a side product of rapid industrial development of human society. Plants, due to their sessile nature, should adapt to these challenging genotoxic environmental conditions and develop resistance. Herein, we evaluated the response of three natural ecotypes of Arabidopsis thaliana (L.) Heynh (Oasis, Columbia-0, and Chernobyl-07) to cadmium, using discovery gel-based proteomics. These accessions are differing by level of tolerance to heavy metal probably achieved by various exposure to chronic ionizing radiation. Based on the pairwise comparison (control versus cadmium-treated) we recognized 5.8-13.4% of identified proteins as significantly altered at the presence of cadmium. Although the majority of photosynthesis-related proteins were found to be less abundant in all ecotypes it was noted that in contrast to the sensitive variants (Col and Oas), the tolerant Che accession may activate the mechanism preserving photosynthesis and energy production. Also, proteins modulating energy budget through alternative route and mediating higher resistance to heavy metals were upregulated in this ecotype. Although we suggest that regulation of enzymes acting in peptide and protein synthesis, protection of the plants against various abiotic stresses, or those neutralizing the effects of reactive oxygen species are rather associated with general response to cadmium, they were found to be altered more intensively in the Che accession. Thus, the identified affected proteins may represent good candidate molecules for molecular breeding to improve tolerance of crops to heavy metal stress.


Assuntos
Arabidopsis/fisiologia , Cádmio/metabolismo , Ecótipo , Poluentes Ambientais/metabolismo , Estresse Fisiológico , Adaptação Fisiológica/efeitos da radiação , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Proteômica , Exposição à Radiação , Especificidade da Espécie
6.
J Proteome Res ; 17(9): 3114-3127, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30084641

RESUMO

Plum pox virus (PPV, family Potyviridae) is one of the most important viral pathogens of Prunus spp. causing considerable damage to stone-fruit industry worldwide. Among the PPV strains identified so far, only PPV-C, PPV-CR, and PPV-CV are able to infect cherries under natural conditions. Herein, we evaluated the pathogenic potential of two viral isolates in herbaceous host Nicotiana benthamiana. Significantly higher accumulation of PPV capsid protein in tobacco leaves infected with PPV-CR (RU-30sc isolate) was detected in contrast to PPV-C (BY-101 isolate). This result correlated well with the symptoms observed in the infected plants. To further explore the host response upon viral infection at the molecular level, a comprehensive proteomic profiling was performed. Using reverse-phase ultra-high-performance liquid chromatography followed by label-free mass spectrometry quantification, we identified 38 unique plant proteins as significantly altered due to the infection. Notably, the abundances of photosynthesis-related proteins, mainly from the Calvin-Benson cycle, were found more aggressively affected in plants infected with PPV-CR isolate than those of PPV-C. This observation was accompanied by a significant reduction in the amount of photosynthetic pigments extracted from the leaves of PPV-CR infected plants. Shifts in the abundance of proteins that are involved in stimulation of photosynthetic capacity, modification of amino acid, and carbohydrate metabolism may affect plant growth and initiate energy formation via gluconeogenesis in PPV infected N. benthamiana. Furthermore, we suggest that the higher accumulation of H2O2 in PPV-CR infected leaves plays a crucial role in plant defense and development by activating the glutathione synthesis.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Nicotiana/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Vírus Eruptivo da Ameixa/patogenicidade , Carotenoides/biossíntese , Clorofila/biossíntese , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Metabolismo Energético/genética , Genótipo , Glutationa/biossíntese , Proteínas de Choque Térmico/classificação , Proteínas de Choque Térmico/metabolismo , Interações Hospedeiro-Patógeno/genética , Peróxido de Hidrogênio/metabolismo , Espectrometria de Massas , Oxirredução , Fotossíntese/genética , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Vírus Eruptivo da Ameixa/classificação , Vírus Eruptivo da Ameixa/genética , Vírus Eruptivo da Ameixa/crescimento & desenvolvimento , Prunus avium/virologia , Prunus domestica/virologia , Nicotiana/metabolismo , Nicotiana/virologia
7.
J Cell Mol Med ; 22(1): 695-699, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28782909

RESUMO

We report the silencing of CA1 mRNA in PC3 and MDA cells. The levels of mRNA coding CA1 protein in the knock-down mRNA (CA1 siRNA) cells have been measured by RT-PCR and were approximately 5% (PC3) and 20% (MDA-MB-231), respectively, of the level of control (Mock siRNA) used during silencing. In PC3 and MDA-MB-231 cells, the mRNAs for COL1A1 and COL4A4 were up-regulated. The mRNAs for CTHRC1, LAMC2, and WNT7B were not changed when compared to the control. The morphology of the cells during the treatments remained the same. On the Western blots, the lysate from the silenced cells showed lower levels of CA I as well.


Assuntos
Anidrases Carbônicas/genética , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias/enzimologia , Neoplasias/genética , Anidrases Carbônicas/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Glycobiology ; 28(6): 382-391, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29548035

RESUMO

Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs. Herein, we provide a comprehensive overview of the biochemical and structural abnormalities in pancreatic tissue from two male FD patients with blood group B. In both patients, we found major accumulation of a variety of complex B-GSLs carrying predominantly hexa- and hepta-saccharide structures. The subcellular pathology was dominated by deposits containing B-glycoconjugates and autofluorescent ceroid. The contribution of Gb3Cer to the storage was minor. This abnormal storage pattern was specific for the pancreatic acinar epithelial cells. Other pancreatic cell types including those of islets of Langerhans were affected much less or not at all.Altogether, we provide evidence for a key role of B-antigens in the biochemical and morphological pathology of the exocrine pancreas in FD patients with blood group B. We believe that our findings will trigger further studies aimed at assessing the potential pancreatic dysfunction in this disease.


Assuntos
Doença de Fabry/metabolismo , Glicoesfingolipídeos/metabolismo , Pâncreas/metabolismo , Sistema ABO de Grupos Sanguíneos/metabolismo , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Estudos de Casos e Controles , Doença de Fabry/sangue , Doença de Fabry/patologia , Galactose/análise , Galactose/metabolismo , Glicoesfingolipídeos/química , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Masculino , Pessoa de Meia-Idade , Pâncreas/ultraestrutura
9.
Appl Microbiol Biotechnol ; 102(23): 10231-10244, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30259098

RESUMO

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.


Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Genes Bacterianos , Piperidonas/metabolismo , Streptomyces/genética , Sequência de Aminoácidos , Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Marcadores Genéticos , Microbiologia Industrial , Família Multigênica , Plasmídeos/genética , Plasmídeos/metabolismo , Plicamicina/análogos & derivados , Plicamicina/biossíntese , Streptomyces/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo
10.
Molecules ; 23(9)2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30189595

RESUMO

Cyclodextrins (CDs) were used in the present study for the ring-opening oligomerization (ROO) of l-lactide (LA) in order to synthesize biodegradable products with possible applications in pharmaceutical and medical fields. The practical importance of ROO reactions may reside in the possibility of synthesizing novel CD derivatives with high purity due to the dual role played by CDs, the role of the initiator through the hydroxylic groups, and the role of the catalyst by monomer inclusion in the CD cavity. The analyzed compounds were CDs modified with oligolactides obtained through ROO reactions of l-lactide in dimethylformamide. The resulting CD isomeric mixtures were investigated using classical characterization techniques such as gel permeation chromatography and nuclear magnetic resonance. Moreover, advanced mass spectrometry (MS) techniques were employed for the determination of the average number of monomer units attached to the cyclodextrin and the architecture of the derivatives (if the monomer units were attached as a single chain or as multiple chains). Thus, fragmentation studies effectuated on two different instruments (ESI Q-TOF and MALDI TOF) allowed us to correlate the size of the oligolactide chains attached to the CD with the observed fragmentation patterns.


Assuntos
Ciclodextrinas/química , Ésteres , Espectrometria de Massas , Polimerização , Polímeros , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Parasitol Res ; 116(11): 3087-3095, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28975409

RESUMO

Wild-living rodents are important hosts for zoonotic pathogens. Bartonella infections are widespread in rodents; however, in Slovakia, knowledge on the prevalence of these bacteria in small mammals is limited. We investigated the prevalence and diversity of Bartonella species in the spleens of 640 rodents of six species (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus, and Micromys minutus) and in the European mole (Talpa europaea) from three different habitat types in south-western and central Slovakia. Overall, the prevalence of Bartonella spp. in rodents was 64.8%; a rate of 73.8% was found in natural habitat (deciduous forest), 56.0% in suburban forest park and 64.9% in rural habitat. Bartonella spp. were detected in 63.0% of A. flavicollis, 69% of My. glareolus and 61.1% of M. arvalis and in T. europaea. However, Bartonella were not found in the other examined rodents. Molecular analyses of the 16S-23S rRNA intergenic spacer region revealed the presence of four different Bartonella spp. clusters. We identified B. taylorii, B. rochalimae, B. elizabethae, B. grahamii and Bartonella sp. wbs11 in A. flavicollis and My. glareolus. Bartonella genotypes ascribed to B. taylorii and B. rochalimae were found in M. arvalis. B. taylorii was identified in T. europaea. Questing Ixodes ricinus ticks that were collected at the study sites were not infected with Bartonella. This study improves our understanding of the ecoepidemiology of Bartonella spp. in Europe and underlines the necessity for further research on Bartonella-host-vector associations and their consequences on animal and human health in Slovakia.


Assuntos
Arvicolinae/microbiologia , Infecções por Bartonella/epidemiologia , Infecções por Bartonella/microbiologia , Bartonella , Murinae/microbiologia , Animais , Bartonella/classificação , Bartonella/genética , Bartonella/isolamento & purificação , DNA Intergênico/genética , Vetores de Doenças , Europa (Continente) , Feminino , Humanos , Ixodes/microbiologia , Masculino , Prevalência , Eslováquia/epidemiologia
12.
Gen Physiol Biophys ; 36(4): 361-371, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28653649

RESUMO

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.


Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Mutação de Sentido Incorreto/genética , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/enzimologia , Laranja de Acridina , Sequência de Aminoácidos , Ativação Enzimática , Estabilidade Enzimática , Células Hep G2 , Humanos , Relação Estrutura-Atividade
13.
Beilstein J Org Chem ; 13: 779-792, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28546834

RESUMO

Biodegradable oligolactide derivatives based on α-, ß- and γ-cyclodextrins (CDs) were synthesized by a green procedure in which CDs play the role of both the initiator and the catalyst. The synthetic procedure in which CDs and L-lactide (L-LA) are reacting in bulk at relatively high temperature of 110 °C was investigated considering the structural composition of the products. The obtained products were thoroughly characterized via mass spectrometry methods with soft ionization like matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). Liquid chromatography (LC) separation with evaporative light scattering detection (ELSD) and NMR analysis were employed in order to elucidate the structural profiles of the obtained mixtures. The results clearly demonstrate that the cyclodextrins were tethered with more than one short oligolactate chain per CD molecule, predominantly at the methylene group, through ring opening of L-LA initiated by primary OH groups.

15.
Proteomics ; 14(16): 1868-81, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24909302

RESUMO

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.


Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Coxiella burnetii/química , Proteômica/métodos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Cromatografia Líquida , Humanos , Octoxinol , Polietilenoglicóis/química , Febre Q/microbiologia , Espectrometria de Massas em Tandem
16.
J Exp Bot ; 65(9): 2335-50, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24648569

RESUMO

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.


Assuntos
Arabidopsis/metabolismo , Medicago sativa/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Ativação Enzimática , Expressão Gênica , Medicago sativa/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Transporte Proteico , Sais/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo
17.
Gen Physiol Biophys ; 33(1): 137-44, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24448368

RESUMO

Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.


Assuntos
Apoptose , Diterpenos/química , Regulação Neoplásica da Expressão Gênica , Fenantrenos/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/química , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Compostos de Epóxi/química , Compostos Macrocíclicos/química , Microscopia de Fluorescência , NF-kappa B/metabolismo , Norepinefrina/química , Análise de Sequência com Séries de Oligonucleotídeos , Oxazóis/química , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Tapsigargina/química
18.
Microorganisms ; 12(4)2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38674697

RESUMO

In this study, we conducted an extensive investigation of the biodegradation capabilities and stress response of the newly isolated strain Pseudomonas veronii SM-20 in order, to assess its potential for bioremediation of sites contaminated with polycyclic aromatic hydrocarbons (PAHs). Initially, phenotype microarray technology demonstrated the strain's proficiency in utilizing various carbon sources and its resistance to certain stressors. Genomic analysis has identified numerous genes involved in aromatic hydrocarbon metabolism. Biodegradation assay analyzed the depletion of phenanthrene (PHE) when it was added as a sole carbon and energy source. We found that P. veronii strain SM-20 degraded approximately 25% of PHE over a 30-day period, starting with an initial concentration of 600 µg/mL, while being utilized for growth. The degradation process involved PHE oxidation to an unstable arene oxide and 9,10-phenanthrenequinone, followed by ring-cleavage. Comparative proteomics provided a comprehensive understanding of how the entire proteome responded to PHE exposure, revealing the strain's adaptation in terms of aromatic metabolism, surface properties, and defense mechanism. In conclusion, our findings shed light on the promising attributes of P. veronii SM-20 and offer valuable insights for the use of P. veronii species in environmental restoration efforts targeting PAH-impacted sites.

19.
J Proteome Res ; 12(11): 4862-9, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24007624

RESUMO

The amount of clinically relevant, allergy-related proteins in wheat grain is still largely unknown. The application of proteomics may create a platform not only for identification and characterization, but also for quantitation of these proteins. The aim of this study was to evaluate the data-independent quantitative mass spectrometry (MS(E)) approach in combination with 76 wheat allergenic sequences downloaded from the AllergenOnline database ( www.allergenonline.org ) as a starting point. Alcohol soluble extracts of gliadin and glutenin proteins were analyzed. This approach has resulted in identification and quantification of 15 allergenic protein isoforms that belong to amylase/trypsin inhibitors, γ-gliadins, and high or low molecular weight glutenins. Additionally, several peptides carrying four previously discovered epitopes of γ-gliadin B precursor have been detected. These data were validated against the UniProt database, which contained 11764 Triticeae protein sequences. The identified allergens are discussed in relation to Baker's asthma, food allergy, wheat dependent exercise induced anaphylaxis, atopic dermatitis, and celiac disease (i.e., gluten-sensitive enteropathy). In summary, the results showed that the MS(E) approach is suitable for quantitative analysis and allergens profiling in wheat varieties and/or other food matrices.


Assuntos
Epitopos/genética , Extratos Vegetais/genética , Proteínas de Plantas/metabolismo , Triticum/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Bases de Dados Genéticas , Espectrometria de Massas/métodos , Proteínas de Plantas/genética , Proteômica
20.
J Proteome Res ; 12(11): 4799-806, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24111740

RESUMO

Starting in 2007, we have grown soybean (Glycine max [L.] Merr. variety Soniachna) and flax (Linum usitatissimum, L. variety Kyivskyi) in the radio-contaminated Chernobyl area and analyzed the seed proteomes. In the second-generation flax seeds, we detected a 12% increase in oil content. To characterize the bases for this increase, seed development has been studied. Flax seeds were harvested in biological triplicate at 2, 4, and 6 weeks after flowering and at maturity from plants grown in nonradioactive and radio-contaminated plots in the Chernobyl area for two generations. Quantitative proteomic analyses based on 2-D gel electrophoresis (2-DE) allowed us to establish developmental profiles for 199 2-DE spots in both plots, out of which 79 were reliably identified by tandem mass spectrometry. The data suggest a statistically significant increased abundance of proteins associated with pyruvate biosynthesis via cytoplasmic glycolysis, L-malate decarboxylation, isocitrate dehydrogenation, and ethanol oxidation to acetaldehyde in early stages of seed development. This was followed by statistically significant increased abundance of ketoacyl-[acylcarrier protein] synthase I related to condensation of malonyl-ACP with elongating fatty acid chains. On the basis of these and previous data, we propose a preliminary model for plant adaptation to growth in a radio-contaminated environment. One aspect of the model suggests that changes in carbon assimilation and fatty acid biosynthesis are an integral part of plant adaptation.


Assuntos
Adaptação Biológica/efeitos da radiação , Acidente Nuclear de Chernobyl , Linho/genética , Linho/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Modelos Biológicos , Proteoma/efeitos da radiação , Adaptação Biológica/genética , Carbono/metabolismo , Radioisótopos de Césio/análise , Eletroforese em Gel Bidimensional , Linho/química , Regulação da Expressão Gênica de Plantas/genética , Glicólise , Óleo de Semente do Linho/análise , Proteoma/genética , Ácido Pirúvico/metabolismo , Sementes/química , Sementes/crescimento & desenvolvimento , Sementes/efeitos da radiação , Solo/química , Radioisótopos de Estrôncio/análise , Espectrometria de Massas em Tandem , Ucrânia
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